Patent Application
20190183856
The present invention relates to a method of treating cancer. In particular, the present invention relates to a method of treating colon cancer.
Cancer is a disease involving abnormal cell growth with the potential to invade or spread to other areas of the body. Colon cancer is one of the most common human tumors in developed and developing countries. It is estimated that 1.361 million people were diagnosed with and 0.694 million people died of colon cancer worldwide in 2012.
In view of the demand for effectively treating cancer, particularly colon cancer, improvements in method are desired.
One example embodiment is a method of treating a cancer in a patient in need thereof. The method includes administering a therapeutically effective amount of a compound to the patient to treat the cancer, the compound is represented by Formula I:
Another example embodiment is a method of treating cancer in a patient in need thereof. The method includes diagnosing the patient having cancer associated p53 mutation; classifying the patient as Group 1 if the diagnosis of p53 mutation is positive, and classifying the patient as Group 2 if the diagnosis of p53 mutation is negative; and administering a therapeutically effective amount of a compound to the patient in Group 2 to treat the cancer, and the compound is represented by Formula I.
Other example embodiments are discussed herein.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Example embodiments relate to methods of treating a cancer in a patient in need thereof. The methods comprise administering a therapeutically effective amount of a compound of [3-Amino-5-[(2,6-dimethylphenyl)amino]-4-(phenylsulfonyl)-2-thienyl](4-fluorophenyl)methanone (APTM). APTM is represented by below Formula I.
Example embodiments relate to methods of treating cancer in a patient in need thereof. The methods comprise diagnosing if the patient having cancer associated p53 mutation; classifying the patient as Group 1 if the diagnosis of p53 mutation is positive, and classifying the patient as Group 2 if the diagnosis of p53 mutation is negative; administering a therapeutically effective amount of a compound to the patient in Group 2 to treat the cancer, and the compound is represented by Formula I.
Example embodiments relate to a pharmaceutical composition comprising a compound and a pharmaceutically acceptable excipient. The compound is represented by Formula I.
In one example embodiment, the cancer is colon cancer. In another example embodiment, the patient does not has p53 mutation. In a further embodiment for example, the p53 mutation is deletion of p53.
[3-Amino-5-[(2,6-dimethylphenyl)amino]-4-(phenylsulfonyl)-2-thienyl](4-fluorophenyl)methanone (APTM) is a synthesized thiophene heterocyclic compound with a high activity and novel structure.
APTM was dissolved in dimethylsulfoxide (DMSO) and stored at −40° C. until use. Sulforhodamine B (SRB), trichloroacetic acid (TCA), crystal violet and Hoechst 33258 were obtained from Sigma Aldrich. McCoy's 5A (Modified) medium, fetal bovine serum (FBS), TrypLE™ Express enzyme and penicillin-streptomycin (10,000 U/mL) were purchased from GIBCO. Annexin V: FITC Apoptosis Detection Kit I was purchased from BD Biosciences and cell cycle detection kit was purchased from Nanjing Key GEN BioTECH. The primary antibodies for cleaved nuclear poly (ADP-ribose) polymerase (cPARP), p53 and Bax were purchased from Cell Signaling. β-actin was purchased from Sigma. Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Inc.
Human colon cancer cell line HCT116 (p53+/+) was purchased from ATCC and the p53−/− HCT116 cell line was provided by Bert Vogelstein, Johns Hopkins University. Cells were cultured in McCoy's 5A medium supplemented with 10% FBS, 100 units/ml penicillin G, and 100 μg/ml streptomycin in humidified atmosphere with 5% CO2. Cells were passaged twice weekly to maintain logarithmic growth.
The anti-proliferative effects of APTM on cancer cell lines were assessed by sulforhodamine B (SRB) colorimetric assay. Cells were seeded in 96-well plates at densities of 5,000 cells per well and cultured overnight. Then cells were susceptible to APTM at indicated concentrations and cultured for indicated times. After incubation, attached cells were fixed with 50 μL cold 50% (w/v) trichloroacetic acid (TCA) for 1 hour at 4° C., washed 5 times with slow-running tap water, and stained with 100 μL 0.4% (w/v) SRB. Optical density at 515 nm (OD515) was measured using a microplate reader (Molecular Devices) after mixing the protein-bound dye with 200 μL 10 mM Tris base solution (pH 10.5). The relative cell growth rate was determined with the following equation: Relative Growth (%)=OD (treated)/OD (control). The IC50 value was defined as the concentration required for a 50% reduction in cell growth.
HCT116 cells were plated at 3,000 cells/well in six-well plates and cultured with indicated concentrations of APTM for 8 days. Cells were stained with 0.2% (w/v) crystal violet in buffered formalin for 20 minutes, and colonies were then photographed and quantified as previously described.
Cell cycle distribution was determined by staining DNA with propidium iodide (PI). Briefly, 1.0×106 cells were incubated with or without APTM for 48 hours. Cells were washed with cold PBS and fixed in 70% ethanol at −20° C. for 2 hours. After washing with PBS, cells were stained with cold PI solution (20 μg/ml PI and 200 μg/mI RNase in PBS) for 30 minutes at room temperature in the dark. The percentage of cells in different phases of the cell cycle was determined by flow cytometer (BD Bioscience) and analyzed using FlowJo software.
Annexin V-FITC/PI double staining method was employed for the apoptosis assay in HCT116 p53+/+ and HCT116 p53−/− cells (1×106/well, 6-well plate). After 48 hours of treatment with APTM, cells were harvested in 15 ml centrifuge tubes by gentle scraping followed by centrifugation at 300×g for 5 minutes at room temperature. Cell suspension was washed two times with cold PBS by centrifugation at 300×g for 5 minutes at room temperature. Then cells were harvested, washed twice with cold PBS, and resuspended in 1×binding buffer (100 μL). Cells were transferred into a 1.5 ml micro-centrifuge tubes and stained with propidium iodide (5 μL) and FITC annexin V (5 μL). Cells were briefly vortexed after incubation for 15 minutes in the dark at room temperature. Then cells were filtered and analyzed by flow cytometry. Total apoptotic cells (FITC to Annexin V positive) were counted.
Cells were plated in 6-well plates (200,000 cells/well) and treated with indicated concentrations of APTM. After incubation for 48 hours, cells were collected, washed with PBS and stained with Hoechst 33258 (11.1 g/ml) in buffered formalin solution containing 5.6% NP-40. Apoptotic and living cells were viewed through DAPI filter of fluorescence inverted microscope (Leica DM2500 Fluorescence Microscope) at 400× magnifications.
HCT116 cells were treated with APTM at indicated concentrations for 48 hours, and harvested via trypsinization. Protein samples were prepared by scratching cells in RIPA buffer containing protease inhibitor cocktail (Roche) and diluted in SDS-PAGE protein sample buffer. Samples were heated for 5 minutes at 100° C. Protein concentrations were measured using Direct Detect® Infrared Spectrometer (Millipore, USA) according to the manufacturer's instructions. Equal amount of proteins were loaded on 4-20% SDS-polyacrylamide (PAGE) gel. After electrophoresis, gels were transferred to a PVDF membrane (Millipore) and incubated with primary antibodies overnight at 4° C. The membranes were then washed with TBST and incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies (1:10000, Santa Cruz, Calif., USA) for 45 min at room temperature. Proteins were visualized with SuperSignal West Dura Extended Duration Substrate or SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) using the Amersham Imager 600 western blotting system.
The in vitro effect of APTM on the proliferation of human colon cancer HCT116 cells and the growth inhibition effect thereof are studied. By employing the p53 deficient HCT116 cells, it was found that induction of apoptosis by APTM is p53 dependent.
The proliferative effect of APTM on human colon cancer cell line HCT116 was examined using SRB assay. HCT116 cells were treated with a series of APTM concentrations of 0.33 μM, 3.33 μM and 33.3 μM for 48 hours. As shown in
2. APTM does not Inhibit Cell Cycle Progression in HCT116 Cells
Cell cycle arrest is a key intracellular event contributing to reduced cell proliferation. It was checked whether APTM could alter cell cycle progression on colon cancer cells. Cell cycle distribution of HCT116 cells treated with APTM was analyzed by flow cytometry. As shown in
To further elucidate whether the growth inhibitory activity of APTM resulted from induction of apoptosis, HCT116 cells treated with 0, 3, 10 and 30 μM of APTM for 48 hours were stained with Hoechst and visualized under a fluorescent microscope. As shown in
The results were further demonstrated by a concentration-dependently induction of cleaved nuclear poly(ADP-ribose) polymerase (cPARP) with APTM treatment as shown in
To test whether the effect of APTM on apoptosis induction and cell proliferation inhibition is dependent on p53, an isogenic HCT116 cell line lacking p53 (HCT116 p53−/−) is employed for further studies. Comparing with wild type HCT116 cells (HCT116 p53+/+), which showed apoptotic morphological changes with APTM treatment (as shown in
Under physiologic conditions, p53 is negatively regulated by E3 ubiquitin ligase MDM2 and maintained at low levels. While in response to a wide range of stress stimuli, such as DNA damage, oxidative stress, nutrient deprivation, oncogene expression and hypoxia, the interaction between p53 and MDM2 is perturbed and p53 protein is stabilized. Once stabilized, p53 stand as the “guardian of the genome” through induction of apoptosis, cell cycle arrest, DNA repair and senescence.
The anti-proliferative effect of APTM on colon cancer HCT116 cells with an IC50 value of 6.57 μM is studied. The growth inhibitory effect of APTM on colon cancer cells is confirmed by time course experiment and colony formation assay. It further shows that APTM induces p53 protein concentration-dependently in HCT116 p53+/+ cells.
One of the most remarkable functions of p53 is the induction of apoptosis. This occurs through either the extrinsic death receptor pathway, transcription dependent intrinsic mitochondrial pathway, or transcription independent cytosolic pathway. The pro-apoptotic protein Bax was induced with p53 protein following APTM treatment (as shown in
Block 501 states determine a patient with cancer.
In one example embodiment, the cancer is colon cancer. In another example embodiment, the patient does not has p53 mutation. In a further embodiment for example, the p53 mutation is deletion of p53. In another example embodiment, the colon cancer with deletion of p53 can be determined using commercially available methods.
Block 502 states administer the compound of Formula I to the patient to treat the cancer.
In one example embodiment, the compound is administered directly or in pharmaceutical compositions along with suitable carriers or excipients. In one example embodiment, suitable routes of administration may, for example, include oral, rectal, transmucosal, nasal, or intestinal administration and parenteral delivery. The compound or the pharmaceutical composition that includes the compound can be administered locally. For example, the compound can be delivered via injection or in a targeted drug delivery system, such as a depot or sustained release Formulation.
Block 601 states diagnose the patient having cancer associated p53 mutation. In an example embodiment, the p53 mutation is deletion of p53.
Block 602 states classify the patient as Group 1 if the diagnosis of p53 mutation is positive, and classify the patient as Group 2 if the diagnosis of p53 mutation is negative.
Block 603 states administer a therapeutically effective amount of the compound represented by Formula I to the patient in Group 2.
In one example embodiment, the compound is administered directly or in pharmaceutical compositions along with suitable carriers or excipients. In one example embodiment, suitable routes of administration may, for example, include oral, rectal, transmucosal, nasal, or intestinal administration and parenteral delivery. The compound or the pharmaceutical composition that includes the compound can be administered locally. For example, the compound can be delivered via injection or in a targeted drug delivery system, such as a depot or sustained release Formulation.
As used herein, the term “patient” encompasses mammals and non-mammals. Examples of mammals include, but are not limited to, humans, chimpanzees, apes monkeys, cattle, horses, sheep, goats, swine; rabbits, dogs, cats, rats, mice, guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish and the like.
As used herein, the term “therapeutically effective amount” refers to any amount of a compound which, as compared to a corresponding patient who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder.
As used herein, the term “treat,” “treating” or “treatment” refers to methods of alleviating, abating or ameliorating a disease or condition symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition either prophylactically and/or therapeutically.
As used herein, the term “administration” or “administering” of the patient compound refers to providing a compound of an example embodiment and/or prodrugs thereof to a patient in need of treatment.
As used herein and in the claims, “comprising” means including the following elements but not excluding others.
